Treatment for toxoplasmosis with a composition comprising a lincosamide and a spiropiperidyl derivative of rifamycik S

ABSTRACT

A method of reducing the severity of toxoplasmosis resulting from infection of a patient with Toxoplasma gondii by administering to a patient in need of such treatment, either after infection or before exposure to infection, a therapeutically effective amount of a compound that is a spiropiperidyl derivative of rifamycin S, wherein the derivative comprises an imidazole ring that includes carbons at positions 3 and 4 of the rifamycin ring, the carbon at position 2 of the imidazole ring also being a ring carbon at position 4 of a piperidine ring system, thereby forming a spiropiperidyl ring system, the spiropiperidyl ring system optionally comprising a lower hydrocarbon substituent on the nitrogen of the piperidine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 08/203,539, filedFeb. 28, 1994, now U.S. Pat. No. 5,529,994 which is acontinuation-in-part of application Ser. No. 08/057,288, filed May 5,1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the field of disease treatment andprophylaxis. More particularly it relates to the treatment andprophylaxis of Toxoplasma gondii infections.

2. Description of the Background

Toxoplasmosis is caused by the parasitic protozoan, Toxoplasma gondii.In humans, the disease is traditionally associated with the developingfetus in whom it can cause severe neurological problems manifesting ashydrocephaly, mental retardation and/or blindness [1, 2]. In healthyadults, the disease is typically mild, producing few if any symptoms. Inimmunocompromised adults, however, the parasite can cause severe or evenfatal disease [3, 4, 5]. The disease also occurs in other mammals and isa leading cause of spontaneous abortion in sheep.

The parasite itself is extremely widespread and is typically acquiredthrough the ingestion of undercooked meat in which tissue cystscontaining the parasite may reside. This form is highly infectious ifthe meat is not well cooked. Alternatively, the parasite can becontracted through ingestion of foods contaminated with oocysts that areshed in the feces of infected cats. The oocyst is the product of thecomplete sexual cycle. The oocyst form is highly resistant todestruction by natural elements and can persist in the soil for morethan one year after excretion by the cat. In the U.S.A., serologicalstudies indicate that about 10-50% of the population has had contactwith the parasite, the prevalence depending on the geographic localesand ethnic group [21]. In countries where eating lightly cooked or rawmeat is more common, this figure can rise to as much as 85% (e.g., inFrance [7]). The incidence of disease in the developing fetus is,fortunately, not as high as these figures might at first suggest becauseit appears that the fetuses of women who are infected for a significantperiod of time prior to becoming pregnant are generally not at risk[21].

Diagnosis of congenital infection has in the past relied on serology(reviewed in [1,21]). This can be done postnatally or, ideally,pre-natally and relies on the relative titers of IgG and IgM (to deducewhether the titers are due to a current infection or legacy of a pastinfection). The factors contributing to the severity of disease in thedeveloping fetus have been poorly understood. The only well-establishedfactor is that the time of initial infection of the mother relative toconception is critical: infection significantly before conception suchthat an effective immune response has been mounted by the mother,results in little if any fetal disease. Infection immediately before orafter conception (i.e., in the first trimester of pregnancy) results insevere disease for about 10-15% of fetuses [21].

In the past two decades, toxoplasmosis has dramatically increased in arelatively new group of patients who are in some way immunodeficient asa result of post-transplantation therapy [5, 9, 10, 22], neoplasticdisease [11, 12, 13, 22] or, most recently, acquired immunodeficiencysyndrome (AIDS) [3, 4, 5]. In such immunodeficient patients, theparasite can cause a disseminated, potentially fatal form of the disease[5, 22, 27].

Typical AIDS patients with toxoplasmosis exhibit signs referable to thecentral nervous system as the first symptom of the disease (reviewed in[22]), as one of the tissues most affected by the parasite is the brain,where massive numbers of parasites and of tissue cysts can be found.Infection is not limited to the brain, however, and parasites and tissuecysts can be found throughout the body [11]. The typical routine fordiagnosis includes serology, computed tomography, magnetic resonanceimaging and/or brain biopsy [1, 15, 16]. Of these, the only definitiveroute to diagnosis is the brain biopsy as this enables the directvisualization of the parasite, using immunoperoxidase staining [17].

In almost all AIDS patients and in most cases of toxoplasmosis in cancerpatients and renal transplant recipients, toxoplasmosis results from arecrudescence of a previous latent (i.e., chronic) Toxoplasma infection.In contrast are patients at risk for the acute acquired infection, suchas the fetus of a previously uninfected pregnant woman or a previouslyuninfected organ transplant recipient who receives an organ from a seropositive (i.e., Toxoplasma-infected) donor.

In general, there are three types of therapy: acute therapy, primaryprophylaxis, and secondary prophylaxis. Acute therapy refers totreatment during an acute phase of an infection. In certain severelyimmunocompromised patient groups, this is followed by secondaryprophylaxis (also known as maintenance therapy), which may be given overthe entire life of a patient. Primary prophylaxis refers to treatmentgiven to prevent the infection from occurring. Primary prophylaxis isoften used in heart transplant recipients who are seronegative and whoreceive a heart from a seropositive donor. Primary prophylaxis is alsoused in pregnant women to prevent transmission from the mother to thefetus; that is, treatment is intended to prevent the mother who acquiredthe acute infection during pregnancy from passing the parasite to herfetus, as well as to treat the fetus in utero. Primary prophylaxis isalso frequently used in AIDS patients to prevent activation of theirlatent (chronic) toxoplasma infections.

The course of treatment for toxoplasmosis in pregnant individuals isdetermined by the stage in pregnancy and whether the infection is acuteor chronic. The purpose of early treatment is to attempt to preventtransmission of the parasite to the fetus. However, the fetus may betreated by treating the mother during gestation. If infection is acute,the antibiotic spiramycin may be administered but is of unprovenefficacy. More effective drugs such as pyrimethamine and sulfadiazine,especially when used in combination, are often used after the firsttrimester of pregnancy (pyrimethamine may be teratogenic) when thediagnosis of infection of the fetus has been established by prenataldiagnostic techniques. Otherwise this particular drug combination isgenerally not used during pregnancy because of the potential toxicityfor the mother and for the developing ferns [21].

Treatment of toxoplasmosis in non-pregnant individuals is initiated andmaintained with a drug regimen involving a combination of folateantagonists, such as pyrimethamine and sulfadiazine [1, 14]. If thedisease is identified soon enough, treatment is reasonably effective incombatting the acute disease. However, due to poor tolerance of thedrugs, especially of the sulfa compounds in AIDS patients, maintenanceon the drug therapy is frequently not possible, and recrudescence of theinfection is often observed (that is, the drug therapy reduces but doesnot eliminate the parasite infection).

Rifamycin compounds are macrocyclic antibiotics that have been shown tobe useful in a number of selective therapeutic applications. For examplerifampin has the following structure: ##STR1## However, rifampin wastested for effectiveness in treating toxoplasmosis and was shown to haveno protective effect in mice challenged with a lethal inoculum oftoxoplasma [18]. Rifampin at concentrations of 50 μg/ml and greatersignificantly inhibited multiplication of toxoplasma in L-cell cultures.However, similar concentrations also inhibited growth of L-cells.Because the toxicity of rifampin for L-cells and its inhibition ofToxoplasma multiplication intracellularly in vitro occurred at the sameconcentration of the drug, it was reported [18] that rifampin was likelyto inhibit Toxoplasma multiplication by its toxic effect on the L-cells.On the other hand, rifamycin compounds are generally considered to beeffective against a limited number of pathological organisms, generallyGram-positive bacteria (including mycobacteria, staphylococci, andstreptococci) and some Gram-negative bacteria (e.g., Brucella,Chlamydia, Haemophilus, Legionella, and Neisseria. spp); otherGram-negative bacteria (e.g., enterobacteria) are less sensitive, andspirochaetes and mycoplasma are known to be insensitive to treatment[19]. Individual compounds within the rifamycin series can be quitespecific in their clinical indications. For example, the Physicians DeskReference (1993 edition) lists only tuberculosis and asymptomaticinfection with N. meningitidis as indications for treatment withrifadin. The related compound rifabutin, which is a spiropiperidylrifamycin derivative, has also primarily been used in treatingmycobacterial infections, notably tuberculosis [20]. More recently ithas been used for primary prophylaxis of Mycobacteriumavium-intracellulare infections in patients with AIDS [25]. However,these uses have been directed to bacteria, not protozoans which aremembers of the animal kingdom).

Accordingly, there remains a need for the development of therapeutic andprophylactic methods that can measurably add to the reliability ofdisease reduction in toxoplasmosis. By adding to the spectrum of drugsavailable for treating toxoplasmosis, problems arising from parasiteresistance and side effects of existing medications can be overcome.

LITERATURE CITED.

1. McCabe, R. E. and Remington, J. S. (1983). Eur. J. Clin. Micro. 2:95-104.

2. Dubey, J. P. and Beatty C. P. In Toxoplasmosis in animals and man,CRC Press, Boca Raton, Fla., U.S.A., pp. 1-220, 1988.

3. Gransden, W. R. and Brown, P. M. (1983). Brit. Med. J. 286: 6378.

4. Ensberger, W., Helm, E. B., Hopp, G., Stille, W. and Fischer, P.-.A.(1985). Deutsche Med. Wochenschrift 110: 83-86.

5. Luft, B. J., Brooks, R. G., Conley, F. K., McCabe, R. E. andRemington, J. S. (1984). JAMA 252: 913-917.

6. Feldman, H. A. (1965). Amer. J. Epidemiol. 81: 385-391.

7. Desmonts, G. and Couvreur, J. (1974). N. Engl. J. Med. 290:1110-1116.

8. Remington, J. S. and Desmonts, G. (1976). In J. S. Remington and J.O. Klein (eds): Infectious Diseases of the Fetus and Newborn Infant.Philadelphia: Saunders, p. 191.

9. Peacock, J. E. J. r., Folds, J., Orringer, E., Luft, B. and Cohen, M.S. (1983). Arch. Intern. Med. 143: 1235-1237.

10. Cohen, S. N. (1970). J. Am. Med. Assn. 211: 657-660.

11. Gleason, T. H. and Hamlin, W. B. (1974). Arch. Intern. Med. 134:1059-1062.

12. Vietzke, W. M., Gelderman, A. H., Grimley, P. M. and Valsamis, M. P.(1968). Cancer 21: 816-827.

13. Frenkel, J. K., Nelson, B. M. and Arias-Stella, J. (1975). Hum.Path. 6: 97-111.

14. Krahenbuhl, J. L. and Remington, J. S. (1982). In S. Cohen and K. S.Warren (eds): Immunology of Parasitic Infections. Oxford: Blackwell, pp.356-421.

15. Cesbron, J. Y., Capron, A., Oviaque, G. and Santoro, F. (1985). J.Imm. Meth. 83: 151-158.

16. Erlich, H. A., Rodgers, G., Vaillancourt, P., Araujo, F. G. andRemington, J. S. (1983). Infect. Immun. 41: 683-690.

17. Conley, F. K., Jenkins, K. A. and Remington, J. S. (1981). Hum.Pathol. 12: 690-698.

18. Remington, J. S., Yagura, T., and Robinson, W. S. (1970). The effectof rifampin on Toxoplasma gondii. Proc. Soc. Exp. Biol. and Med. 135:167-172.

19. Singleton, P., and Sainsbury, D., eds. (1987). Dictionary ofMicrobiology and Molecular Biology, Second Edition, Antony Rowe, Ltd.,Chippenham (England).

20. Woodley, C. L., and Kilburn, J. O. (1982). Am. Rev. Respir. Dis.126: 586-587.

21. Remington, J. S. and Desmonts, G. (1990). Toxoplasmosis. InInfectious Diseases of the Ferns and Newborn Infant, Third Edition, J.S. Remington, J. O. Klein, eds. Philadelphia: W. B. Saunders Company,pp. 89-195.

22. Israelski, D. M. and Remington, J. S.: AIDS-associatedtoxoplasmosis. In The Medical Management of AIDS, Third Edition, M. A.Sande, and P. A. Volberding, eds. Philadelphia: W. B. Saunders Company,pp. 319-345, 1992.

23. McLeod, R., and Remington, J. S. (1992). Toxoplasmosis. In NelsonTextbook of Pediatrics, Fourteenth Edition, R. E. Behrman, R. M.Kliegman, W. E. Nelson and V. C. Vaughan, eds. Philadelphia: The W. B.Saunders Company, pp. 883-892.

24. McLeod, R. and Remington, J. S. (1987). Toxoplasmosis. In Harrison'sPrinciples of Internal Medicine, Eleventh Edition, E. Braunwald, K. J.Isselbacher, R. G. Petersdorf, J. D. Wilson, J. B. Martin and A. S.Fauci, eds. New York: McGraw-Hill Book Company, pp. 791-797.

25. Hoy, J., Mijch, A., Sandland, M. Grayson, L. Lucas, R., and Dwyer,B. (1990). Quadruple-Drug Therapy for Microbacterium Avium-IntracellularBacteremia in AIDS patients. J. Infect. Dis. 161:801-805.

26. Araujo, F. G., Lin, T., and Remington, J. S. (1993). The Activity ofAtovaquone (566C80) in Murine Toxoplasmosis is Markedly Augmented whenUsed in Combination with Pyrimethamine or Sulfadiazine, J. Infect. Dis.167:494-497.

27. Ruskin, J. and Remington, J. S. (1976). Toxoplasmosis in theCompromised Host. Ann. Intern. Med., 84:193-199, 1976.

28. Araujo, F. G. (1992). Depletion of CD4+ T cells but not inhibitionof the protective activity of IFN-γ prevents cure of toxoplasmosismediated by drug therapy in mice. J. Immunol. 149:3003-3007.

29. Araujo, F. G., R. M. Shepard, and J. S. Remington (1991). In vivoactivity of the macrolide antibiotics azithromycin, roxithromycin andspiramycin against Toxoplasma gondii. Eur. J. Clin. Microbiol. Infect.Dis. 10:519-524.

30. Araujo, F. G., J. Huskinson-Mark, W. E. Gutteridge, J. S. Remington(1992). In vitro and in vivo activities of the hydroxynaphthoquinone566C80 against the cyst form of Toxoplasma gondii. Antimicrob. AgentsChemother. 36:326-330.

31. Araujo, F. G., J. Huskinson, and J. S. Remington (1991). Remarkablein vitro and in vivo activities of the hydroxynaphthoquinone 566C80against tachyzoites and cysts of Toxoplasma gondii. Antimicrob. Agentsand Chemother. 35:293-299.

32. Araujo, F. G., P. Prokocimer, and J. S. Remington (1992).Clarithromycin-minocycline is synergistic in a murine model oftoxoplasmosis. J. Infect. Dis. 165:788.

33. Chiodini. R. J., J. M. Kreeger. and W. R. Thayer (1993). Use ofrifabutin in treatment of systemic Mycobacterium paratuberculosisinfection in mice. Antimicrob. Agents Chemother. 37:1645-1648.

34. Dannemann, B., J. A. McCutcham, D. M. Israelski, D. Antoniskis, C.Leport, B. Luft, J. Nussbaum, N. Clumeck, P. Morlat, J. Chiu, J.-L.Vilde, M. Orellana, D. Feigal, A. Bartok, P. Halsetin, J. Leedom, J. S.Remington and the California Collaborative Treatment Group (1992).Treatment of toxoplasmic encephalitis in patients with AcquiredImmunodeficiency Syndrome: A randomized trial comparing pyrimethamineplus clindamycin to pyrimethamine plus sulfadiazine. Ann. Intern. Med.116:33-43.

35. Dannemann, B. R., D. M. Israelski, and J. S. Remington (1988).Treatment of toxoplasmic encephalitis with intravenous clindamycin.Arch. Intern. Med. 148:2477-2482.

36. Ellner, J. J., M. J. Goldberger, and D. M. Parenti (1991).Mycobacterium avium infection in AIDS: a therapeutic dilemma in rapidevolution. J Infect Dis, 163:1326-1335.

37. Heifets, L. B., M. D. Iseman, and P. J. Lindholm-Levy.Bacteriostatic and bactericidal effects of rifabutin (ansamycin, LM 427)on Mycobacterium avium clinical isolates, p. 180-183. In M. Casal (ed.)Mycobacteria of Clinical Interest 1986. Amsterdam: Elsevier Science.

38. Horsburgh, C. R. (1991). Mycobacterium avium complex infections inthe acquired immunodeficiency syndrome. N. Engl. J. Med. 324:1332-1338.

39. Hughes, W. T., W. Kennedy, J. Shenep, P. M. Flynn, S. V.Hetherington, G. Fullen, D. J. Lancaster, D. S. Stein, S. Palte, D.Rosenbaum, S. H. T. Liao, M. R. Blum, M. D. Rogers (1991). Safety andpharmacokinetics of 566C80, a hydroxynaphthoquinone withanti-Pneumocystis carinii activity: A phase I study in humanimmunodeficiency virus (HIV)-infected men. J. Infect. Dis. 163:843-848.

40. Israelski, D. M., B. R. Dannemann, and J. S. Remington.Toxoplasmosis in patients with AIDS, p. 241-264. In M. A. Sande, and P.A. Volberding (eds.) The medical management of AIDS-1990. Philadelphia:W. B. Sounders.

41. Israelski, D. M., F. G. Araujo, F. K. Conley, Y. Suzuki, S. D.Sharma, J. S. Remington (1989). Treatment with anti-L3T4 (CD4)monoclonal antibody reduces the inflammatory response in toxoplasmicencephalitis. J. Immunol. 142:954-958.

42. Narang, P. K., R. C. Lewis, and J. R. Bianchine (1992). Rifabutinabsorption in humans: Relative bioavailability and food effect. Clin.Pharmac. Therap. 52:335-341.

43. McCabe, R. E. and J. S. Remington. Toxoplasma gondii, p. 2090-2103.In Third ed. Principles and Practice of Infectious Diseases-1990. G. L.Mandell, J. Dauglas R. G., and J. E. Bennett (eds.) London: ChurchillLivingstone, Inc.

44. Hunter, C. A., J. S. Abrams, M. H. Beaman, and J. S. Remington(1993). Cytokine mRNA in the central nervous system of SCID miceinfected with Toxoplasma gondii: importance of T-cell independentregulation of resistance to T. gondii. Infect. Immun. 61:4038-4044.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide a class ofcompounds effective in protecting an infected or potentially infectedhost mammal against toxoplasmosis.

It is a further object of invention to provide such a class of compoundswith a minimum number of side effects.

It is a still further object of the invention to provide a compositioneffective in vivo in preventing the lethal effects of toxoplasmosis orotherwise reducing the severity of the disease, preferably with a classof compounds of proven clinical tolerance for other indications.

These and other objects of the invention, as will hereafter become morereadily apparent, have been accomplished by providing a method ofreducing the severity of toxoplasmosis resulting from infection of amammalian host with Toxoplasma gondii, which comprises administering toa mammalian host in need of such treatment, either after infection orbefore exposure to such infection, a therapeutically effective amount ofa compound that is a spiropiperidyl derivative of rifamycin S, whereinthe derivative comprises an imidazole ring that includes carbons atpositions 3 and 4 of the rifamycin ring, the carbon at position 2 of theimidazole ring also being a ring carbon at position 4 of a piperidinering system, thereby forming a spiropiperidyl ring system, thespiropiperidyl ring system optionally comprising a lower hydrocarbonsubstituent on the nitrogen of the piperidine.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention now being generally described, the same will be betterunderstood by reference to the following detailed description ofspecific embodiments when considered in combination with the figuresthat form part of the specification, wherein:

FIG. 1A is a graph showing survival of mice infected with T. gondii andtreated with various concentrations of rifabutin (RIF).

FIG. 1B is a graph showing survival of mice following infection withcysts of the C56 strain of T. gondii and treated with variousconcentrations of rifabutin (RIF).

FIG. 2 is a graph showing survival of mice infected with T. gondii andtreated with low concentrations of rifabutin (RIF) alone or incombination with sulfadiazine (SUL).

FIG. 3 is a graph showing survival of mice infected with T. gondii andtreated with moderate concentrations of rifabutin alone or incombination with sulfadiazine.

FIG. 4 is a graph showing survival of mice infected with T. gondii andtreated with high concentrations of rifabutin alone or in combinationwith sulfadiazine.

FIG. 5 is a graph showing survival of mice infected with T. gondii andtreated with rifabutin (RIF) alone or in combination with pyrimethamine(PYR).

FIG. 6 is a graph showing survival of mice infected with T. gondii andtreated with rifabutin (RIF) alone or in combination with clindamycin(CLINDA).

FIGS. 7A and 7B depict survival of mice infected with T. gondii andtreated with rifabutin (RIF) alone or in combination with atovaquone(ATO).

FIG. 8 is a graph showing survival of mice infected with T. gondii andtreated with rifabutin (RIF) alone or in combination with azithromycin(AZITHRO).

FIG. 9 is a graph showing survival of mice infected with T. gondii andtreated with rifabutin (RIF) alone or in combination withclarithromycinin (CLARI).

DESCRIPTION OF SPECIFIC EMBODIMENTS

The present inventors have discovered that spiropiperidyl derivatives ofrifamycin S are surprisingly effective in protecting a mammalian hostagainst infection by the disease-causing organism associated withtoxoplasmosis. This discovery contrasts with previously availableinformation on the effectiveness of rifamycin compounds, such asrifampin, which was previously shown to be ineffective in protectingagainst toxoplasmosis. This remit is particularly surprising in view ofthe relatively close structure of the ineffective rifampin compound andthe spiropiperidyl derivatives of rifamycin S that are now shown to beeffective.

Compounds of the invention are semi-synthetic derivatives of rifamycin Scomprising a fused imidazole ring that includes carbons at positions 3and 4 of the rifamycin ring, the carbon at position 2 of the imidazolering also being a ring carbon at position 4 of a piperidine ring system,thereby forming a spiropiperidyl ring system. The nitrogen atom of thepiperidine ring is optionally substituted with a lower hydrocarbonsubstituent, typically containing from 1 to 8 (preferably 3-5) carbons,usually branched, and most preferably being an iso-butyl group. However,other hydrocarbon groups can be present at this location, such asmethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl,iso-pentyl, hexyl, and iso-hexyl. Unsaturated analogues of thesesaturated alkyl groups (especially alkenes, particularly those in whichthe double bond (or bonds) is located so that an sp³ hybridized carbonis attached to the nitrogen atom of the piperidine ring) are alsopermitted.

Accordingly, the compounds used in the practice of the invention willtypically have the formula: ##STR2## in which R represents one of theindicated alkyl groups.

These compounds do not represent a new class of compounds but rather arecompounds that have been previously known for other uses. Thespiropiperidyl derivatives of rifamycin S are semi-synthetic moleculesproduced by modification of natural rifamycins produced by Nocardiamediterranei (also known as Streptomyces mediterranei). Mixtures ofrifamycins A-E are generally produced in culture, but the proportion ofthe desired intermediate rifamycin B can be increased by the addition ofsodium diethyl barbiturate in the growth medium. Rifamycin B is only anintermediate, since an aqueous oxygenated solutions spontaneously givesrise to other rifamycins, such as O and S. Rifamycin S is the startingpoint for the production of a number of semi-synthetic rifamycins. Theseinclude the specific compound known by the name rifamycin (also referredto as rifamycin SV), which is obtained by mild reduction of rifamycin S.Other derivatives produced by modification of rifamycin S includerifamide and rifampicin (also called rifampin), the latter having astructure as shown in the background section above. Total synthesis ofrifamycin S has also been reported. See, for example, H. Nagaoka, etal., J Am. Chem. Soc. 102:7962 (1980); H. Iio, et al., ibid. 7965; H.Nagaoka and Y. Kishi, Tetrahedron, 37:3873 (1981). For a review ofchemistry of rifamycins, see K. L. Reinhart, et al., Fortschr. Chem.Org. Naturst. 33:231-307 (1976). Synthesis of spiropiperidyl rifamycinsis described in L. Marsili, et al., J. Antibiot. 34:1033-1038 (1981).Spiropiperidyl rifamycin compounds and their activity as antibacterialsagainst Gram-positive and Gram-negative bacteria, includingMicrobacterium tuberculosis, are described in U.S. Pat. No. 4,219,478.Also see related U.S. Pat. Nos. 4,086,225, and 4,327,096. As an exampleof the systematic chemical nomenclature of compounds of the invention,rifabutin is (9S,12E,14S,15R,16S,17R,18R,19R,20S ,21S,22E,24Z)-6,16,18,20-tetrahydroxy-1'-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethyl-spiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-furo[2',3',7,8]naphth[1,2-d]imidazole-2,4'-piperidine]-5,10,26-(3H,9H)-trione-16-acetate.

Compounds of the invention method are soluble in chloroform andmethanol, sparingly soluble in ethanol, and slightly soluble in water.The compounds can be prepared in standard pharmaceutical compositions ofthe same type used for other rifamycin compounds. A composition for usein vivo generally will contain a pharmaceutically acceptable carrier. Bythis is intended either solid or liquid material, which may be inorganicor organic and of synthetic or natural origin, with which the activecomponent of the composition is mixed or formulated to facilitateadministration to a subject. Any other materials customarily employed informulating pharmaceutical are suitable. Solid carriers include naturaland synthetic cloisonne silicates, for example natural silicates such asdiatomaceous earths; magnesium silicates, for example talcs; magnesiumaluminum silicates, for example attapulgites and vermiculites; aluminumsilicates, for example kaolinites, montmorillonites, and micas; calciumcarbonate; calcium sulfate; synthetic hydrated silicone oxides andsynthetic calcium or aluminum silicates; elements such as carbon orsulfur; natural and synthetic resins such as polyvinyl alcohol; andwaxes such as paraffin and beeswax. Examples of suitable liquid carriersinclude water and aqueous solutions containing oxygenated organiccompounds such as ethanol. Buffers and other materials normally presentin pharmaceutical preparations, such as flavoring and suspending agents,can also be present. Pharmaceutical carriers differ from typicalsolutions and suspensions in that they are specifically prepared for usein vivo to exclude substances that may be harmful to the host to whomthe composition is administered (e.g., removal of bacterial toxins).

As an example of a pharmaceutical composition, rifabutin can be presentat 150 mg per capsule in a gelatin capsule intended for oraladministration. The remainder of the capsule will contain, for example,as inactive ingredients, microcrystalline cellulose, magnesium stearate,red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, andedible white ink.

Compositions containing spiropiperidyl rifamycins have been indicatedfor other pharmaceutical uses, such as anti-mycobacterial indications,including tuberculosis. The clinical pharmacology of these compounds isthus known. For example, following a single oral admission of 300 mg ofrifabutin in normal subjects, rifabutin is readily absorbed from thegastrointestinal tract with mean (±SD) peak plasma levels (C_(max)) of375 (±267) mg/mL (range: 141 to 1033 mg/mL) attained in 3.3 (±0.9) hours(T_(max) range: two to four hours). Plasma concentrations post-C_(max)decline in an apparent bi-phasic manner. Kinetic dose-proportionalityhas been shown in healthy normal volunteers and in early symptomaticHuman Immunodeficiency Virus (HIV) positive patients at doses from 300mg to 900 mg. Rifabutin is eliminated slowly from plasma in the samemanner as other rifamycin compounds.

As indicated previously, the specific method of the present invention isdirected to the treatment and/or prophylaxis of toxoplasmosis. Fortreatment, the dosage on the first day (referred to as a loading dose)is often but not necessarily higher than on succeeding days. A typicalloading dose would be 0.25-400 mg/kg/day (preferably 1-200, morepreferably 2-200), with typical treatment dosages at half these values.These doses are in the range of normal doses for other rifamycincompounds. For prophylactic use, doses in the lower half of the normalrange are used, generally without a loading dose.

In a typical treatment regimen, spiropiperidyl rifamycins can beadministered to humans as a single dose of about 300 mg per os ("po";orally) once a day, generally at breakfast, or, if not tolerated in asingle dose, can be divided into two doses of about 150 mg each withmorning and evening meals. The half-life in serum of rifabutin is 16hours. The drug is eliminated in part by the kidneys, with urineconcentrations being about 100-fold higher than those in the plasma. Thedrug also appears in the bile at a concentration similar to that in theurine. The drug is taken up by all tissues and is especiallyconcentrated in lungs, where levels reach 5-10 times higher than thosein plasma. For a study of the clinical effects of rifabutin, see R. J.O'Brien et al., Reviews of Infectious Diseases, 9:519-530 (1987).

Subjects to be treated include those acutely infected with Toxoplasmagondii and, for prophylactic use, those subjects who may in the futurebe exposed to the disease-causing organism or immunosuppressed patientswith chronic toxoplasma infection to prevent recrudescence or relapse ofthe infection. In view of the widespread occurrence of the organism,this can include all immunocompromised individuals. Selection of aparticular dose and determination of the timing of dosages is best leftto the decision of a physician or veterinarian in the same manner as forother treatments with rifamycin compounds, so that the dosage can beraised or lowered as necessary. Although the method is intended for usein humans in part, veterinary use is also contemplated, particularly indomesticated mammals (such as sheep, pigs, and cats) that are also knownhosts for T. gondii.

A particularly interesting aspect of the spiropiperidyl rifamycincompounds is their ability to act synergistically with sulfonamides.Sulfonamides comprise a well known group of antimicrobial agents thatare mostly derivatives of sulfanilamide (p-aminobenzenesulfonamide).They halt or retard the growth of a wide range of Gram-positive andGram-negative bacteria, as well as various protozoa (such as coccidiaand Plasmodium spp). Sulfanilamides are often used in combination withother chemotherapeutic agents for treating urinary tract infections andvarious intestinal disorders.

Organisms which synthesize their own folic acid and which cannot use anexogenous supply of the vitamin are sensitive to sulfonamides providedthat the cells are permeable to the drug. This is a result of theability of sulfonamides to act as structural analogs of p-aminobenzoicacids (PABA). Sulfonamides competitively inhibit the incorporation ofPABA during folic acid synthesis. Thus, the combinations in which theyare most often used involve other folic acid antagonists. Organismswhich require exogenous folic acid for growth are insensitive tosulfonamide treatment.

There are many sulfonamide drugs that differ in their clinicalproperties and toxicities. Most are derivatives bearing substituents atthe nitrogen of the sulfonamide group (i.e., NH₂ C₆ H₄ SO₂ NHR, where Rrepresents the substituent). Substitution at the p-amino group normallyresults in loss of antibacterial activity. However, such derivatives areoften hydrolyzed in vivo to an active form and can therefore beadministered in inactive form. For example, p-N-succinylsulfathiazoleand phthalylsulfathiazole are inactive but are hydrolyzed in the lowerintestine to release the active component sulfathiazole.

A number of active sulfonamides include, e.g., sulfacetamide(N-[4-aminophenyl)sulfonyl]-acetamide); sulfadiazine; sulfadimethoxine(4-amino-N-(2,6-dimethoxy-4-pyrimidinyl)benzenesulfonamide);sulfadimidine (sulfamethazine:4-amino-N-(4.6-dimethyl-2-pyrimidinyl)benzenesulfonamide);sulfaguanidine (4-amino-N-(aminoiminomethyl)benzenesulfonamide);sulfamethoxazole(4-amino-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide);sulfamethoxazole(4-imino-N-(5-methyl-3-isoxazolyl)benzenenesulfonamide); sulfathiazole(4-azino-N-2-thiaamide); and sulfathiazole(4-amino-N-2-thiazolylbenzenesulfonamide).

The present inventors have discovered that in addition to thespiropiperidyl rifamycin compounds' synergistic interaction withsulfonamides, the spiropiperidyl rifamycin compounds also actsynergistically as demonstrated herein with a number of antimicrobialcompounds in the treatment of toxoplasmosis, including theantibacterials clindamycin, clarithromycin, and azithromycin; theanti-malarial drug pyrimethamine; and atovaquone. These synergisticinteractions provide a further advantage within the general scope of theinvention (treatment with spiropiperidyl rifamycins, whether or not incombination with other drugs) by providing certain synergistic effectswhen spiropiperidyl rifamycins are used in combination with selectedother classes of drugs.

Accordingly, one embodiment of the invention is the use ofspiropiperidyl rifamycin compounds in combination with lincosamides toachieve a synergistic effect. Lincosamides comprise a well known groupof antimicrobial agents that contain lincosamine (i.e.,6-amino-6,8-dideoxyoctose). They halt or retard the growth of a range ofGram-positive and Gram-negative bacteria. There are many lincosamidedrugs that differ in their clinical properties and toxicities. Anylincosamide can be used in combination with rifabutin or otherspiropiperidyl rifamycin compounds in the treatment or prevention oftoxoplasmosis; preferred are those that further act synergistically astaught herein. Active lincosamides include lincomycin, its semisyntheticderivative clindamycin (7-chloro-7-deoxylincomycin), and celesticetin.Other useful lincosamide derivatives include those that can behydrolyzed in vivo to an active form and can therefore be administeredin inactive form. For example, clindamycin palmitate HCl is inactive invitro, but rapid in vivo hydrolysis converts it to antibacteriallyactive clindamycin. Clindamycin phosphate, a water soluble ester ofclindamycin and phosphoric acid, is another preferred lincomycinderivative.

A number of individual compounds and class of compounds actsynergistically with spiropiperidyl rifamycin compounds. For example,spiropiperidyl rifamycin compounds act synergistically withpyrimethamine. Pyrimethamine is a folic acid antagonist effectiveagainst protozoans. Spiropiperidyl rifamycin compounds also actsynergistically with hydroxynaphthoquinones. A preferredhydroxynaphthoquinone is atovaquone. Other preferredhydroxynapththoquinone antibiotics are those that have activity againstT. gondii when used alone. Spiropiperidyl rifamycin compounds actsynergistically with azalides. Preferred azalides are azithromycin andits derivatives. Other preferred azalide antibiotics are those that haveactivity against T. gondii when used alone. Spiropiperidyl rifamycincompounds further act synergistically with macrolide antibiotics.Preferred macrolides are clarithromycin and its derivatives. Otherpreferred macrolide antibiotics are those that have activity against T.gondii when used alone. Such preferred macrolides include roxithromycinand spiramycin. Macrolides of the invention also include angolamycin,carbomycin, chalcomycin, cirramucin, erythromycin, lankomycin,leucomycin, megalomycin, methymycin, narbomycin, niddamycin,oleandomycin, relomycin, troleandomycin, tylosin, polyene antibiotics,and their active derivatives.

These and the other antimicrobial agents discussed herein may be natural(produced from natural or genetically engineered sources),semi-synthetic or wholly synthetic. As these are all known compounds,descriptions of synthesis can be found in the published scientificliterature.

In one aspect of the invention, these drugs are used in combination withrifabutin or other spiropiperidyl rifamycin compounds as pharmaceuticalsor to produce medicaments useful in the treatment or prevention oftoxoplasmosis infection or toxoplasmosic encephalitis ("TE"). Apharmaceutical would comprise a therapeutically effective amount ofrifabutin or other spiropiperidyl rifamycin compound in combination witha therapeutically effective amount of one or more of the drugs discussedherein as synergistically effective against toxoplasma. Alternatively,these drugs can be used individually to produce medicaments that arethen used in combination with rifabutin or other spiropiperidylrifamycin compounds to treat or prevent toxoplasmosis infection or TE.Combination medicaments or treatments comprise rifabutin or otherspiropiperidyl rifamycin compounds with at least one otheranti-toxoplasma compound as taught herein. Pharmaceutical carriers,dosages, routes of administration, and treatment regimens are aspreviously discussed herein, although they can be effectively modifiedby a clinician, using as a guideline the dosages presented herein anddosages previously known for use in treatment of toxoplasmosis. Ofcourse, because of the synergistic activity discovered herein, lowerdosages than those previously reported are now available to theclinician or veterinarian.

In addition to their synergistic interaction with the above compounds,the spiropiperidyl rifamycin compounds should also act synergisticallywith a number of antibiotic compounds in the treatment of toxoplasmosisincluding antiviral such as gamma interferon, compounds effectiveagainst T. gondii such as macrolides roxithromycin and spiramycin andtetracylines, e.g. minocycline, and their congeners, and other compoundseffective against other protozoan-caused diseases.

One specific aspect of the invention within the scope of treating"toxoplasmosis" is a method of reducing the severity of toxoplasmosicencephalitis resulting from infection of a mammalian host withToxoplasma gondii. TE can be reduced in severity by administering to ahost in need of treatment, either after infection or before exposure toa T. gondii infection, a therapeutically effective amount of a compoundthat is a spiropiperidyl derivative of rifamycin S, wherein thederivative includes an imidazole ring that includes carbons at positions3 and 4 of the rifamycin ring, the carbon at position 2 of the imidazolering also being a ring carbon at position 4 of a piperidine ring system,thereby forming a spiropiperidyl ring system, the spiropiperidyl ringsystem optionally including a lower hydrocarbon substituent on thenitrogen of the piperidine. A preferred compound for treatment isrifabutin. Pharmaceutical carriers, dosages, routes of administration,and treatment regimens are as previously discussed herein for treatmentof toxoplasmosis. In a further aspect of the invention, TE can bereduced in severity by administering a therapeutically effective amountof a combination of a spiropiperidyl rifamycin compound with a drugdiscussed herein that enhances the effectiveness a spiropiperidylrifamycin compound for treatment or prevention of toxoplasmosis.Pharmaceutical carriers, dosages, routes of administration, andtreatment regimens are as previously discussed herein for combinationdrug treatment of toxoplasmosis.

As provided herein, and as demonstrated by the following Examples,spiropiperidyl rifamycin derivatives, particularly rifabutin, areeffective in the protection of a mammalian host, including animmunocompromised host, against infection with Toxoplasma gondii. Thecompounds also demonstrate a synergistic effect in combination withother drugs, particularly with sulfonamides, and provide full protectionin this model study against toxoplasmosis. The in vivo results withrifabutin presented in Example 1, which demonstrate that rifabutin aloneis effective in the treatment of toxoplasma related diseases, contrastwith and are unexpected in light of results obtained with rifampin [18].Demonstrated herein is that rifabutin alone is effective in thetreatment of toxoplasma related diseases, and further that rifabutinacts synergistically in combination with other drugs. This addssignificantly to the spectrum of drugs available for treatingtoxoplasmosis, thereby overcoming problems arising from parasiteresistance and side effects of existing medications. In addition,rifabutin was found to decrease the inflammatory response associatedwith toxoplasmic encephalitis (Example 8). Accordingly, the resultspresented herein provide for therapeutic and prophylactic methods thatmeasurably add to the reliability of disease reduction in toxoplasmainfections.

The anti-T. gondii effect in mice with disseminated acute toxoplasmosisdue to infection with RH tachyzoites was enhanced significantly whenrifabutin was used in combination with pyrimethamine, sulfadiazine,clindamycin, or atovaquone, as demonstrated in the Examples. These drugsare preferred since they are either presently in use or are undergoingclinical trials for treatment of toxoplasmosis. Azithromycin orclarithromycin in combination with rifabutin were also demonstrated inthe Examples as enhancing the anti-T. gondii effect. The presentinvention allows for effectively treating toxoplasmosis and toxoplasmicencephalitis in severely immunocompromised patients. As demonstratedherein, the combination of ineffective doses of rifabutin andclindamycin resulted in remarkable and significant protection of micewith disseminated acute toxoplasmosis (Example 4) and in T.gondii-infected immunocompromised mice (Example 9). Clindamycin iscurrently most frequently used as an alternative to sulfonamides whenside-effects develop during treatment with the pyrimethamine-sulfonamidecombination [34, 35]. Furthermore, although pyrimethamine andsulfonamides may be responsible for inconvenient side effects,particularly in AIDS patients, the combinations taught herein allow forsignificant reduction in their dosing to minimize or eliminate theiruntoward side effects. The combination of rifabutin-atovaquone wasdemonstrated as effective against toxoplasmosis (Example 5). Atovaquoneis active both against T. gondii [31, 32] and Pneumocystis carinii [39].Rifabutin, on the other hand, is active against mycobacteria and is nowbeing used for treatment and prophylaxis of organisms of theMycobacterium avium complex [36]. Thus, the results with the combinationrifabutin-atovaquone indicate use of this combination for treatment andprophylaxis of the three of the most frequently encounteredopportunistic agents in AIDS patients.

The invention now being fully described, the following examples areprovided for illustration only and are not intended to limiting of theinvention unless so stated.

EXAMPLES Example 1 Activity of rifabutin against T. gondii in murinemodel of acute toxoplasmosis

An experiment was carried out to determine the in vivo protectiveactivity of rifabutin against toxoplasmosis. The model used was a mousemodel using Swiss Webster female mice weighing approximately 20 grams atthe time of the experiment. The rifabutin used in the experiment wasobtained commercially from Adria as a pharmacologically pure compoundand was solubilized in phosphate buffered saline, pH 7.2, and thensonicated for four pulses of one minute each. The experiment was carriedout on four groups of mice (3 experimental and one control) with 10 miceper group. The mice were infected with T. gondii tachyzoites of the RHstrain obtained from the peritoneal fluid of carrier mice according topublished standard procedures for this model [26]. The peritoneal fluidwas collected into Earle's balanced salt solution containing 10% fetalcalf serum and 10 units of heparin/ml. The preparation was then filteredthrough glass wool, centrifuged at 500×g for 5 minutes at 4° C., and thesediment was resuspended and forced through a 27-gauge needle. Theorganisms were quantified by counting on a Neubauer-Levy hemacytometer,and appropriate dilutions in Eagle's minimum essential medium (MEM) weremade. The lethal inoculum of the RH strain of T. gondii is approximatelyone tachyzoite. Mice infected with our RH strain never survive with anyliving parasites. Thus, survivors in our studies had the organismeradicated by the therapy. Mice were infected with an intraperitonealdose of 2.5×10³ tachyzoites of the RH strain (a dose that normallyresults in death of 100% of normal mice by day 8 or 9). Beginning 24hours after ip infection, one group of mice was treated with 50mg/kg/day of rifabutin, a second with 100 mg/kg/day, and a third with200 mg/kg/day administered orally by gavage for a period of ten days.Thereafter, mice were followed for an additional 20 days and the date ofdeath noted. Mice dying during the experiment were examined for thepresence of T. gondii in their peritoneal fluid for verification of thecause of death.

As shown in FIG. 1A, significant protection was noted in mice treatedwith doses of rifabutin of 100 mg/kg/day and higher. Administration of200 mg/kg/day resulted in survival of 9 out of the 10 mice in that groupfor the entire 30-day test period. In the 100 mg/kg/day group, six micewere still alive 15 days after infection, while two survived the 30-dayobservation period. In contrast, all 10 of the control mice died withinthe expected 9-day period, and only 4 mice survived beyond 10 days inthe 50 mg/kg/day group, with all of the latter dying within 14 days.Thus, the dose of 50 mg/kg/day resulted in significant prolongation oftime to death as compared with untreated controls even though all of themice eventually died. One hundred percent of mice infected ip withtachyzoites of the RH strain survived when treated with doses of 300 or400 mg/kg/day rifabutin. Identical results were obtained when rifabutinwas prepared by dissolution in phosphate buffered saline, pH 6.8,followed by sonication for 30 seconds.

Activity of different doses of rifabutin (RIF) in disseminated acutemurine toxoplasmosis following ip infection with cysts of the C56 strainof T. gondii was also examined. Adult, outbred Swiss-Webster (SW),females, weighing 20 g at the beginning of each experiment were used[26, 29, 30, 31]. Treatment was initiated 3 days after infection.Treatment was for 10 days. Rifabutin was prepared by dissolution inphosphate buffered saline, pH 6.8, followed by sonication for 30seconds. As shown in FIG. 1B, significant protection was obtained formice treated with doses of rifabutin of 100 mg/kg/day and higher.Administration of 200 mg/kg/day resulted in survival of 9 out of the 10mice in that group for the entire 30-day test period. Administration of300 mg/kg/day resulted in survival of 10 out of the 10 mice in thatgroup for the entire 30-day test period. In the 100 mg/kg/day group, sixmice were still alive 15 days after infection, while two survived the30-day observation period. All 10 of the control mice died within theexpected 9-day period, while 4 mice survived beyond 10 days in the 50mg/kg/day group, with all of the latter dying within 14 days. Ten of 10infected mice survived for the entire test period when treated with 300mg/kg/day.

The above results indicate significant activity for rifabutin intreatment of disseminated acute murine toxoplasmosis caused by differentstrains of T. gondii. A dose of 200 mg/kg/day administered for 10 daysprotected at least 80% of the mice against death due to infection eitherwith tachyzoites of the RH strain or with cysts of the C56 strain. Dosesof 300 or 400 mg/kg/day protected 100% of the mice and apparently werenot toxic for the animals since non-infected mice treated with thesedoses did not show any clinical signs of toxicity and did not loseweight. Although pharmacokinetic studies were not conducted in thepresent investigation, in normal human volunteers [42] rifabutin israpidly but incompletely absorbed from the gastrointestinal tract. Theaverage terminal half-life in humans after oral administration was 36hours and the bioavailability was 84.8% [42].

RH tachyzoites and C56 cysts were used in these experiments because ofprevious reports that demonstrated variation in susceptibility ofdifferent strains of T. gondii to different drugs [29]. In addition, thepathogenesis of the infection produced by inoculation of tachyzoites orcysts differ. Ip inoculation of RH tachyzoites results in a fulminantinfection with large numbers of tachyzoites being produced in theperitoneal cavity. One hundred percent mortality usually occurs within 5to 8 days after infection with an inoculum as small as 10² organisms. Incontrast, oral inoculation of cysts of the C56 strain results in aninfection which progresses slower. Depending on the inoculum size,mortality does not occur until approximately 15 days of infection.

Example 2 In vivo activity of rifabutin in combination with asulfonamide against T. gondii in a murine model of acute toxoplasmosis

An experiment was carried out in the manner described for Example 1, butwith the additional administration of a sulfonamide. Conditions were asdescribed in the first example except for the following changes. Sevengroups of mice (10 mice per group) were infected by intraperitonealinjection of 2.5×10³ tachyzoites of T. gondii and then given oraltreatment by gavage with rifabutin beginning 24 hours after infectionand continuing for 10 days. Sulfadiazine, sodium salt, was obtained fromSigma Chemical Co. or City Chem. Corp., New York, N.Y., and wasadministered ad libitum during the entire period of treatment in thedrinking water at a concentration of 80 mg/liter to some groups asfollows:

Group 1--Rifabutin alone, 50 mg/kg/day.

Group 2--Rifabutin alone, 100 mg/kg/day.

Group 3--Rifabutin alone, 200 mg/kg/day.

Group 4--Sulfadiazine alone at 80 mg/L.

Group 5--Rifabutin, 50 mg/kg/day, plus sulfadiazine at 80 mg/L.

Group 6--Rifabutin, 100 mg/kg/day, plus sulfadiazine at 80 mg/L.

Group 7--Rifabutin, 200 mg/kg/day, plus sulfadiazine at 80 mg/L.

Groups 1, 2 and 3 are similar to the three experimental groups discussedand set forth separately in Example 1 and FIG. 1A. For the controlgroup, see Example 1. Results of the experiment are set out in FIGS.2-4. FIG. 2 shows the results of treatment with low concentrations ofrifabutin alone, sulfadiazine alone, or the combination of the two. Theeffect of sulfadiazine alone was slight, in that only one mouse survivedbeyond the 9-day period of survival for the control animals. This singlemouse died by day 11. The combination of a low dose rifabutin andsulfadiazine was not as effective as the low dose of rifabutin byitself. Only one animal survived beyond day 9 for the combination, withthat animal dying on day 13. Four animals survived beyond day 9 forrifabutin alone, the last animal dying on day 14.

Higher doses of rifabutin provided greater protection, as shown in FIGS.3 and 4. There appeared to be a synergistic effect when a dose ofrifabutin of 100 mg/kg/day was used in combination with a dose ofsulfadiazine of 80 mg/liter (FIG. 3). Six animals survived beyond 9 dayswhen treated with rifabutin alone at a dose of 100 mg/kg/day and 2animals survived for the entire 30-day period. Surprisingly, 8 of the 10animals treated with the combination survived beyond 10 days and 6 ofthem survived for the entire 30-day test period (FIG. 3). As shown inFIG. 4, rifabutin at 200 mg/kg/day was highly effective, andsulfadiazine added to that effectiveness. Nine of the 10 animalssurvived the entire test period, with rifabutin alone at thisconcentration, while all 10 survived on the combination.

Example 3 In vivo activity of rifabutin in combination withpyrimethamine against T. gondii in a murine model of acute toxoplasmosis

An experiment was carried out in the manner described for Examples 1 and2, but with the additional administration of pyrimethamine instead of asulfonamide. Pyrimethamine (lot 3F991, Burroughs-Wellcome Co., ResearchTriangle Park, N.C.) was dissolved in a solution of 0.25%carboxymethylcellulose. Conditions were as described in the firstexample except for the following changes. Six groups of mice (5 in thecontrol group, 10 or 15 in the treatment groups as indicated in FIG. 5)were infected by intraperitoneal injection of 2.5×10³ tachyzoites of T.gondii. Treatment with rifabutin alone (50 or 100 mg/kg/day) or incombination with pyrimethamine (10 mg/kg/day) was initiated 24 hoursafter infection and continued for 10 days. Both drugs were administeredorally by gavage as a single daily dose, wither alone or in combination.Test groups were as follows:

Group 1--Control

Group 2--Rifabutin alone, 50 mg/kg/day.

Group 3--Rifabutin alone, 100 mg/kg/day.

Group 4--Pyrimethamine alone, 10 mg/kg/day.

Group 5--Rifabutin, 50 mg/kg/day, plus pyrimethamine, 10 mg/kg/day.

Group 6--Rifabutin, 100 mg/kg/day, plus pyrimethamine, 10 mg/kg/day.

Results of the experiment are set out in FIG. 5. The effects ofrifabutin alone are similar to those seen in the previous examples inwhich average survival rates were extended, with the higher dose givingbetter long-term protection (30-day test period). The combination of alow dose of rifabutin and pyrimethamine gave better protection than foreither drug acting alone. The higher dose of rifabutin in combinationwith pyrimethamine gave the best protection.

Example 4 In vivo activity of rifabutin in combination with clindamycinagainst T. gondii in an acute model of murine toxoplasmosis

Experiments were carried out in the manner described for Examples 1 and3, but with the additional administration of clindamycin instead ofpyrimethamine. Clindamycin (hydrochloride salt, lot 627HJ, Upjohn Co.,Kalamazoo, Mich.) was dissolved in sterile PBS and administered orallyby gavage in a single daily dose of 25 or 50 mg/kg. Conditions were asdescribed in the first example except for the following changes. Fourgroups of mice (5 in the control group, 10 or 15 in the treatment groupsas indicated in FIG. 6) were infected by intraperitoneal injection of2.5×10³ tachyzoites of T. gondii RH strain. Treatment with rifabutin(RIF) alone at 50 mg/kg/day or in combination with clindamycin (CLINDA)at 25 mg/kg/day was initiated 24 hours after infection and continued for10 days. Both drugs were administered orally by gavage as a single dailydose, either alone or in combination.

Results are shown in FIG. 6. The protection afforded by the combinationof rifabutin with clindamycin was remarkable. Ninety percent of miceinfected ip with RH tachyzoites survived the infection when treated witha combination of 50 mg/kg of rifabutin plus 25 mg/kg of clindamycin.These doses of the respective drugs had only slight effects inprotecting mice against death when administered alone. In a secondexperiment, 100% of infected mice treated with the combination survived.

Clindamycin is the drug most frequently used as an alternative tosulfonamides when side-effects develop during treatment with thepyrimethamine-sulfonamide combination [34, 35]. The combination ofineffective doses of rifabutin and clindamycin resulted in a synergisticand significant protection of mice with disseminated acutetoxoplasmosis.

Example 5 In vivo activity of rifabutin in combination with atovaquoneagainst T. gondii in an acute model of murine toxoplasmosis

Experiments were carried out in the manner described for Examples 1 and3, but with the additional administration of atovaquone instead ofpyrimethamine. A stock solution of atovaquone (lot 8810001-158,Burroughs-Welcome, Co.) was prepared in PBS and sonicated for 3 pulsesof 30 seconds each. Working solutions to provide a dose for each mouseof 5 or 10 mg/kg were prepared in 0.25% carboxymethyl-cellulose andsonicated again for one 30-second pulse just prior to administration bygavage. Conditions were as described in the first example except for thefollowing changes. Four groups of mice (5 in the control group, 10 inthe treatment groups as indicated in FIG. 7A and 7B) were infected byintraperitoneal injection of 2.5×10³ tachyzoites of T. gondii RH strain.Treatment with rifabutin (RIF) alone at 100 mg/kg/day or 50 mg/kg/day orin combination with atovaquone (ATO) at 5 mg/kg/day was initiated 24hours after infection and continued for 10 days. Both drugs wereadministered orally by gavage as a single daily dose, either alone or incombination. Control mice were treated with the drug diluent only.

Results are shown in FIGS. 7A and 7B. Treatment with combinations of 50or 100 mg/kg of rifabutin with 5 mg/kg of atovaquone resulted inprotection of mice against death. One hundred mg/kg rifabutin plus 5mg/kg atovaquone protected at least 60% of the mice against death (FIG.7A). Statistical analysis of the data, however, did not revealsignificant differences between the group of mice treated with rifabutinalone and the group treated with the combination. In contrast, when thedose of rifabutin was reduced to 50 mg/kg and used in combination with adose of 5 mg/kg/day of atovaquone, prolongation of time to death wasobtained, and 30% of the mice survived the infection (FIG. 7B). Thedifference between the groups of mice treated with rifabutin alone andthose treated with the combination was statistically significantly(P=0.011).

Since atovaquone is active both against T. gondii [30, 31] andPneumocystis carinii [39], and rifabutin is active against mycobacteria,being used for treatment and prophylaxis of organisms of theMycobacterium avium complex [36], the results presented herein, whichdemonstrate the effectiveness of the combination of rifabutin-atovaquoneagainst T. gondii, provide for the use of this combination for treatmentand prophylaxis of the three of the most frequently encounteredopportunistic agents in AIDS patients.

Example 6 In vivo activity of rifabutin in combination with azithromycinagainst T. gondii in a murine model of acute toxoplasmosis

Experiments were carried out in the manner described for Examples 1 and3, but with the additional administration of azithromycin instead ofpyrimethamine. Conditions were as described in the first example exceptfor the following changes. Four groups of adult Swiss-Webster femalemice (5 in the control group, 5 or 10 in the treatment groups asindicated in FIG. 8) were infected by intraperitoneal injection of2.5×10³ tachyzoites of T. gondii RH strain. Treatment with 50 mg/kg ofazithromycin (AZITHRO) or 50 mg/kg of rifabutin (RIF) alone or incombination was administered using a feeding tube, was initiated 24hours after infection and continued for 10 days. Control mice weretreated with drug diluent only.

Results are shown in FIG. 8. One hundred percent of the mice treatedwith the combination survived at 30 days whereas none of the micetreated with either drug alone survived at 30 days.

Example 7 In vivo activity of rifabutin in combination withclarithromycin against T. gondii in a murine model of acutetoxoplasmosis

Experiments were carried out in the manner described for Examples 1 and3, but with the additional administration of clarithromycin instead ofpyrimethamine. Conditions were as described in the first example exceptfor the following changes. Four groups of adult Swiss-Webster femalemice (5 in the control group, 5 or 10 in the treatment groups asindicated in FIG. 9) were infected by intraperitoneal injection of2.5×10³ tachyzoites of T. gondii RH strain. Treatment with 50 mg/kg ofrifabutin (RIF) or 50 mg/kg of clarithromycin (CLARI) alone or incombination was administered using a feeding tube, was initiated 24hours after infection and continued for 10 days. Control mice weretreated with drug diluent only.

Results are shown in FIG. 9. One hundred percent of the mice treatedwith the combination survived at 30 days whereas 20% of the mice treatedwith clarithromycin alone survived at 30 days.

Example 8 In vivo activity of rifabutin against T. gondii in a murinemodel of toxoplasmic encephalitis ("TE")

Inbred CBA/Ca (CBA) adult females weighing 17 g at the beginning of eachexperiment were used to determine rifabutin activity in treatment oftoxoplasmic encephalitis caused by infection with cysts of the strainME49 of T. gondii [29, 30]. All mice were purchased from SimonsenLaboratories, Gilroy, Calif. and were fed regular laboratory mouse foodand water ad libitum. Each CBA mouse was infected orally with 20 cystsof the ME49 strain [30, 32]. Infected CBA mice were used 5 weeksfollowing infection [37, 39]. Histopathological examination of brains of3 mice euthanized at this time revealed extensive TE with profuseinflammatory infiltrates in the meninges, parenchyma and around smallcapillaries as well as large numbers of cysts of T. gondii. Rifabutinwas dissolved in phosphate buffered saline, pH 6.8, and sonicated for 30seconds. Treatment with 200 mg/kg of rifabutin administered as a singledaily oral dose by gavage was then initiated and continued for 30 days.Five treated and 5 control mice were euthanized by CO₂ narcosis 15 and30 days after initiation of treatment and their brains collected fordetermination of number T. gondii cysts and for histopathologicexamination as previously described [30, 32, 41].

Histopathology of brains of control mice 30 days after infection werecompared with histopathology of brains of rifabutin treated mice 30 daysafter infection. Histopathology of brains of control mice treated withthe diluent only revealed extensive inflammatory exudates in themeninges, parenchyma and around small capillaries as well as numerouscysts of T. gondii. Treatment of infected CBA mice with 200 mg/kg/day ofrifabutin administered alone for 15 days did not significantly reducethe inflammatory response or numbers of T. gondii cysts in brains of the5 treated mice as compared with controls. However, treatment with 200mg/kg/day of rifabutin administered alone for 30 days resulted inremarkable reduction in the inflammatory response in each of the treatedmice when compared to controls. The number of T. gondii cysts in thebrains of the treated mice were not significantly reduced as comparedwith untreated controls.

These results indicate that rifabutin used alone is effective fortreatment of the infection in the central nervous system. Since thenumbers of T. gondii cysts in the brains of treated mice were notsignificantly reduced at the end of the therapy, these results may bedue to a previously unreported, anti-inflammatory activity of rifabutin.Alternatively, rifabutin may act only against free parasites and/oragainst rapidly replicating parasites within cells. The drug may not beable to act against the slowly replicating bradyzoites protected withinthe cyst wall. However, the present invention is not limited by aparticular mechanism of rifabutin action.

Example 9 In vivo activity of rifabutin in combination with clindamycinagainst T. gondii in an immunocompromised mammalian host

Treatment of a T. gondii infected, immunocompromised mammalian hostusing rifabutin in combination with clindamycin was studied.Immunocompromised T- and B-cell deficient SCID ("severe combinedimmunodeficient") mice [44] were infected p.o. with cysts of the ME49strain of T. gondii. ME49 cysts are less virulent than C56 cysts, andaccordingly are more suitable for studies involving immunocompromisedhosts. Clindamycin was dissolved in sterile PBS. Single daily doses ofthe drug combination, 50 mg/kg of clindamycin with 100 mg/kg ofrifabutin, were administered orally by gavage beginning 48 hours afterinfection. SCID controls were treated with carrier solution only. Mousemortality was monitored post infection. At day 25 post infection, 100%of infected, immunocompromised mice receiving daily treatments of thecombination of 100 mg/kg of rifabutin and 50 mg/kg of clindamycinsurvived, whereas 100% of the infected, immunocompromised controls died.This is a synergistic effect since these doses of the respective drugshad no effect in protecting immunocompetent mice against death whenadministered alone under the conditions used in this example. Cessationof the daily drug combination treatment resulted in the death of all theinfected, immunocompromised test animals.

In summary, these examples demonstrate the effectiveness ofspiropiperidyl rifamycin derivatives, particularly rifabutin, in theprotection of a mammalian host, including an immunocompromised host,against infection with Toxoplasma gondii. The compounds also demonstratea synergistic effect in combination with other drugs, particularly withsulfonamides, and provide full protection in this model study againsttoxoplasmosis.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of reducing the severity oftoxoplasmosis resulting from infection of a mammalian host withToxoplasma gondii, which method comprises:administering to a host inneed of said treatment, either after infection or before exposure tosaid infection, a therapeutically effective amount of a lincosamide incombination with a therapeutically effective amount of a compound thatis a spiropiperidyl derivative of rifamycin S, wherein said derivativecomprises an imidazole ring that includes carbons at positions 3 and 4of the rifamycin ring, the carbon at position 2 of said imidazole ringalso being a ring carbon at position 4 of a piperidine ring system,thereby forming a spiropiperidyl ring system, said spiropiperidyl ringsystem optionally comprising a lower hydrocarbon substituent on thenitrogen of said piperidine.
 2. The method of claim 1, wherein saidcompound has a formula ##STR3## wherein R represents a 3-5 carbon alkylgroup.
 3. The method of claim 1, wherein said compound is rifabutin. 4.The method of claim 1, wherein said host is an immunocompromised orpregnant human.
 5. The method of claim 1, wherein said amount is from 1to 200 mg/kg/day.
 6. The method of claim 1, wherein said administeringis by oral ingestion.
 7. The method of claim 1, wherein said lincosamideis clindamycin.